Project Summary (30 lines) Entitled ?Cost Effective, Synergistic Macromolecular Structure Determination, Analysis & Simulation?, this proposal involves systems at the heart of biology: CCT Eukaryote Chaperonin, RNA Polymerase II, and the Ribosome, all of which our colleagues are studying experimentally. We will develop unbiased methods to solve structures with less data. Interested in how molecular machines move as they function, we map out their state-space using multi-scale hybrid methods. All our methods and curated data will be disseminated freely. This project is timely as these macromolecular machines carry out key cellular functions. The tools we develop for structure determination, analysis and simulation will aid others in advancing biomedicine. Michael Levitt, the Principal Investigator has a long career of independent scientific research that started in 1967 when he was one of the first to work in computational biology. His early work set up the conceptual, theoretical and computational framework for protein and DNA structure refinement, structure analysis and macromolecular simulations. He makes computer codes available and continues hands-on software development. He has been productive, scientifically rigorous and impactful for half a century. Particularly innovative is his work for the past five years leading to original methods to both solve biomedically significant structures and simulate functional motion. These areas are continued here by a PI committed to mentoring young scientists as well as engaging in sustained research-community service and public outreach. 1. Develop Novel Approaches to Determination and Refinement of Macromolecular Complexes. The first sub-area will deal with determining the identity of amino acid side chains. The second sub-area will involve a new approach to automatic structure determination requiring no manual intervention. Both methods will be adapted to cryo-EM electron density maps. 2. Develop Novel Approaches to Pathway Analysis of Structures. The first sub-area will deal with structure curation. Essential for ribosome work, it will be increasingly useful as structures accumulate for other macromolecular complexes. The second sub-area focuses on methods to find reaction pathways from multiple structures of such complexes. The third sub-area will test and develop new methods for structure morphing with few degrees of freedom. The forth sub-area will use Molten Zone molecular dynamics to study functional movement. 3. Determine the State-Space of Functional Motion in Chaperonin, RNA Pol II, and the Ribosome. We will identify key states, morph between these states to find reaction paths and run molecular dynamics. Studying these biomedically significant systems in collaboration with experimental colleagues will reveal fascinating details of biology in action. This work will elucidate the relationship between structure and function in large macromolecular machines, a keystone of modern biomedical science.